Hintergrund. Sibutramin ist ein Medikament, das häufig zur Gewichtsreduktion eingesetzt wird. Es hemmt die Wiederaufnahme von Noradrenalin und Serotonin. Noradrenalin-Wiederaufnahme-Hemmung könnte zu arterieller Hypertonie führen. Methoden. In einer doppelt-blinden, randomisierten Cross-Over Studie untersuchten wir 11 junge, gesunde Probanden (7 Männer, 4 Frauen, Alter 27±2 Jahre, BMI 23.1±0.7Kg/m2). Sie nahmen 26 Stunden (h) und 14h vor Studienbeginn 10mg Sibutramin ein, und 2h vor Untersuchungsbeginn 20mg. Die Wirkung verglichen wir mit Placebo und der Kombination von Sibutramin und 200mg des Beta1-Adrenorezeptor-Blockers Metoprolol. Autonome Reflextests, eine graduelle Kipptischuntersuchung, Plasmakatecholamin-Bestimmungen und eine indirekt kalorimetrische Messung wurden durchgeführt. Herzfrequenz- und Blutdruckbestimmungen erfolgten kontinuierlich und nichtinvasiv. Wir zeichneten das Schlagvolumen, das Herzzeitvolumen und den peripheren Widerstand impedanzkardiografisch auf. Ergebnisse. Sibutramin verursachte einen leichten Anstieg der Herzfrequenz in Ruhe (p / Background. Sibutramine, a serotonin and norepinephrine transporter blocker, is widely used as an adjunctive obesitytreatment. Norepinephrine reuptake inhibition with sibutramine conceivably could exacerbate arterial hypertension andpromote cardiovascular disease. Methods. In 11 healthy subjects (7 men, age 27±2 years, body mass index 23.1±0.7 kg/m2), we compared the effect of sibutramine or matching placebo (ingested 26, 14, and 2 hours before testing) on cardiovascular responses to autonomic reflex tests and to a graded head-up tilt test. In addition, we tested sibutramine in combination with metoprolol. Testing was conducted in a double-blind and crossover fashion. Results. Supine systolic blood pressure was 113±3 mm Hg with placebo, 121±3 mm Hg with sibutramine (P

Tumorzellen entwickeln häufig Resistenzen gegen verschiedene strukturell und funktionell unabhängige Zytostatika, was als Multidrug-Resistenz (MDR) bezeichnet wird und die Hauptursache für das Scheitern einer Chemotherapie ist. Mit Hilfe von in vitro-Untersuchungen wurden erhöhte Genexpressionen der ABC-Transporter MDR1, MRP2 und BCRP als maßgebliche Resistenzfaktoren identifiziert, wie z.B. in den Magenkarzinomzellinien EPG85-257RDB (MDR1) und EPG85-257RNOV (BCRP) sowie in der Ovarialkarzinomzellinie A2780RCIS (MRP2). Ziel der Arbeit war die Entwicklung eines auf Ribozym-Technologie basierenden Therapieansatzes, welcher die Expressionen der oben genannten ABC-Transporter simultan inhibiert und dessen Anwendung zur Reversion der zellulären MDR führt. Dazu wurde ein Multitargetmultiribozym (MTMR) entwickelt, das aus in trans-aktiven Ribozymen besteht, die gegen die ABC-Transporter mRNAs gerichtet sind sowie aus in cis-spaltenden Ribozymen und aus Spacer-RNA-Sequenzen. Durch autokatalytische Spaltung in cis konnten die in trans-aktiven Ribozyme aus dem Gesamt-MTMR freigesetzt werden. Die Analyse der kinetischen Parameter des MTMRs ergab, daß die autokatalytisch entstandenen MTMR-Fragmente ihre Substrat-RNAs im Vergleich zu den korrespondierenden Monoribozymen ohne Einbuße an Effizienz spalten können. Darüber hinaus wurde die MTMR-Sequenz stabil in den drei eingangs genannten MDR-Zellinien exprimiert, was in einer signifikanten Reduktion der jeweiligen Ziel-mRNAs (97 % MDR1-, 80 % BCRP-, 96 % MRP2-mRNA) und der entsprechenden Proteine resultierte. Die Multidrug-Resistenz konnte aufgrund der MTMR-Expression um 70% (A2780RCIS), 95% (257RNOV), 100% (257RDB) und die Zytostatikumsakkumulation um 90% (257RNOV-Zellen) sowie 100% (257RDB-Zellen) revertiert werden. Das MTMR stellt erstmalig ein RNA-Konstrukt dar, welches in der Lage ist, simultan mehrere unabhängige Gene funktionell auszuschalten. Es besitzt daher ein großes Potential für zukünftige gentherapeutische Ansätze. / Cancer cells are often insensible against structurally and functionally unrelated drugs that is known as multiple drug resistance (MDR) and the main cause for treatment failure. Overexpression of the ABC-transporters P-gp (ABCB1), MRP2 (ABCC2), and BCRP (ABCG2) is associated with MDR in several cancer cell lines, e.g. in the stomach carcinoma cell lines EPG85-257RDB (P-gp), EPG85-257RNOV (BCRP), and in the ovarian carcinoma cell line A2780RCIS (MRP2). We aimed the development of a novel hammerhead ribozyme-based therapeutic approach capable of simultaneous silencing of the prementioned ABC-transporters, and consequently of reversing MDR phenotypes. We designed a so-called multitarget multiribozyme (MTMR) consisting of trans-acting hammerhead ribozymes directed against the MDR1, MRP2, and BCRP transcripts, of MDR1 homologous spacer sequences, and of cis-acting ribozymes against the spacer sequences. Autocatalytic cleavage in cis excised the full-length MTMR, and released trans-acting hammerhead ribozymes. We also evaluated the catalytic features of the MTMR using large RNA target molecules. Comparison of the kinetic values of the autocatalytically derived MTMR fragments with those of corresponding mono-ribozymes demonstrated an MTMR-mediated substrate cleavage without distinct loss in catalytic efficiency. Moreover, the MTMR was stably expressed in the prementioned multidrug-resistant cancer cell lines, and decreased the targeted transcripts about 97% (MDR1), 80% (MRP2), and 96% (BCRP) as well as the corresponding protein levels, respectively. Cellular MDR could be reverted about 70% (A2780RCIS), 95% (257RNOV), and 100% (257RDB). Additionally, the MTMR reversed mitoxantrone accumulation entirely, and daunorubicin accumulation about 90% in stomach carcinoma cells, respectively. Taken together, the MTMRs capability of simultaneous silencing of multiple genes provides an effective instrument to knockdown genes of interest.

La maladie de Parkinson (MP) provient de la dégénérescence des cellules du locus nigerproduisant de la dopamine. La barrière hémato-encéphalique (BHE) est un véritable obstacle pour le traitement de la MP car elle empêche ou réduit le passage d’un grand nombre de substances pharmacologiques vers le cerveau. L’encapsulation de ces substances dans des liposomes ou des niosomes avant leur libération intra-cérébrale représente une alternative de choix en raison de la biocompatibilité, la biofragmentation, la non-toxicité et les capacités de ciblage de ces systèmes. A l’heure actuelle le traitement de la MP reste un défi, malgré l’existence de nombreux projets de recherche dans ce domaine. Notre hypothèse est que l’administration de pramipexoleencapsulé dans des liposomes et/ou des niosomes pourrait représenter une approche thérapeutique pertinente. Dans le cadre de la thèse, la caractérisation et la cinétique de diffusion des liposomes et niosomescontenant du pramipexole ont été réalisées. La validation de différentes formulations a été réalisée sur un modèle de BHE constitué de co-cultures cellulaires. Les effets du pramipexoleencapsulé dans des liposomes ou desniosomesont ensuite été étudiés dans un modèle de MP chez le rat obtenu par lésion de la voie dopaminergique nigro-striée à l’aide de 6-hydroxydopamine (6-OHDA). Pour cela, nous avons évalué le comportement rotatoire induit par l’amphétamine et l’expression du transporteur de la dopamine (DAT) par autoradiographie quantitative chez des animaux lésés traités ou non par les nanocapsules. Toutes les formulations que nous avons réalisées ont montré une capacité d’encapsulation d’environ 10% pour une taille de 100 nm, avec une cinétique de dispersion compatible avec une utilisation in vivo. Dans notre modèle de co-culture cellulaire, nous avons déterminé que nos formulations permettent le franchissement de la BHE. Chez les animaux lésés à la 6-OHDA, la quantification du DAT indique que l’administration de pramipexole réduit l’intensité de la lésion, que la substance soit administrée seule ou encapsulée dans des niosomes. Ces travaux montrent l’intérêt potentiel de l’administration de principe actif encapsulé pour le traitement de la MP, et devront être poursuivis afin d’optimiser cette approche thérapeutique, notamment au niveau des doses. / Parkinson’s Disease (PD) is degeneration of dopamine producing cells in substantia nigra. Blood-brain barrier (BBB) is a strong obstacle in PD therapy. More penetration and accumulation in the target tissue can be obtained by preventing RES uptake via “stealth effect”. Liposomes and niosomes are the promising systems for being biodegredable, bioavailable, non-toxic and targetable. Although CNS disorders are the first to endorse at their research in the diagnosis and therapy with several framework projects in Europe and over the world, there is still a huge gap in CNS drug delivery and the success of PD therapy. Although different studies have performed with pramipexole, evaluation of penetration and antiparkinsonian effect of pramipexole encapsulated liposomes and niosomes has never been studied before. Among this thesis, nanosized, polyethylene glycol (PEG) coated, neutral and positively charged, pramipexole encapsulated liposomes and noisomes were formulated, characterized and release kinetics of the systems were evaluated. In vitro penetration of all formulations was evaluated in BBB cell co-culture model. Therapeutic efficacy of neutral, pramipexole encapsulated liposomes and niosomes were evaluated in 6-hydroxydopamine (6-OHDA) lesioned rats by rotometer test and autoradiography. All formulations have approximately 10% encapsulation efficiency, around 100 nm particle sizes and fitted to first-order release kinetics. All formulations were found BBB permeable at in vitro cell culture studies. Nanosized, neutral niosomes designated similar but slightly better effect than pramipexole solution in autoradiograhy studies in 6-OHDA lesioned rats. This pramipexole dose is approximately 9 times lesser doses applied with conventional pramipexole tablets for humans in Neurology clinics. Nanosized, pramipexole encapsulated, neutral niosomes showed potential PD therapeutic effect in PD animal model depending on non-ionic surfactant properties of niosomes. / Uzm. Ecz. Mine Silindir Gunay, Parkinson Hastalığı’nın Teşhis ve Tedavisi İçin Kullanılacak Nanoboyutlu Teragnostik İmmünolipozom/İmmunoniozomlar Üzerine İn Vitro İn Vivo Çalışmalar, Hacettepe Üniversitesi – François Rabelais de Tours University, Sağlık Bilimleri Enstitüsü, Radyofarmasi Programı, UMR Inserm U 930, Ekip 3, Moleküler Görüntüleme ve Beyin Programı, Doktora Tezi, Ankara-Tours, 2016. Parkinson Hastalığı (PH) substantia nigra’daki dopamin üreten hücrelerin dejenerasyonundan kaynaklanmaktadır. Kan-beyin bariyeri (KBB) PH’nın tedavisinin önünde kuvvetli bir engeldir. Hedef dokudaki yüksek penetrasyon ve tutulum “stealth etki” ile RES tutulumunun engellenmesi ile sağlanabilir. Lipozom ve niozomlar biyoparçalanırlıkları, biyouyumlulukları, non-toksik ve hedeflendirilebilir olmaları nedeniyle en çok tercih edilen sistemlerdendir. Santral sinir sistemi hastalıklarının araştırılması Avrupa ve tüm dünyada yapılan pekçok çerçeve projelerinde ilk sırada olmasına rağmen, halen beyne ilaç taşınması ve PH’nin tedavi başarısı konusunda büyük boşluklar bulunmaktadır. Pramipeksol ile pek çok çalışma yapılmasına karşılık, bizim çalışmamız pramipeksol enkapsüle edilmiş lipozom ve niozomların beyin penetrasyonunun ve antiparkinson etkisinin değerlendirilmesi konusunda yenidir. Tez kapsamında, nanoboyutlu, PEG kaplı, nötral ve pozitif yüklü lipozom ve niozomların formüle edilmiş, karakterizasyon ve salım kinetikleri değerlendirilmiştir. Tüm formülasyonların KBB geçirgenliği, hücre KBB ko-kültürü çalışmalarında incelenmiştir. Nötral, pramipeksol enkapsüle edilen lipozom ve niozomların tedavi etkinliği in vivo olarak 6-hidroksidopamin (6-OHDA) ile lezyon yapılarak PH modeli oluşturulan sıçanlarda rotametre ve otoradyografi çalışmaları ile incelenmiştir. Tüm formülasyonlar yaklaşık %10 enkapsülasyon etkinliği ve 100 nm civarında partikül boyutu dağılımı ve birinci derece salım kinetiği göstermiştir. Hücre kültürü çalışmalarında, tüm formülasyonların KBB’nden penetre olabildiği saptamıştır. 6-OHDA lezyonlu sıçanlarda Parkinson hastalığının tedavisinde nanoboyutlu, nötral, pramipeksol enkapsüle edilen niozomlar, aynı dozdaki pramipeksol çözeltisi ile benzer hatta biraz daha iyi sonuçlar göstermiştir. Bu doz Nöroloji kliniklerinde Parkinson tedavisinde rutin olarak kullanılan konvansiyonel pramipeksol tabletlerindeki dozun yaklaşık olarak 9 kat düşük dozlarıdır. Nanoboyutlu, pramipeksol enkapsüle edilen, nötral niozomlar, niozomların non-iyonik sürfaktan özellikleri nedeniyle PH modeli sıçanlarda potansiyel bir antiparkinson terapötik etki göstermiştir.

Drug transport across cells and cell membranes in the human body is crucial for the pharmacological effect of drugs. Active transport governed by transport proteins plays an important role in this process. A vast number of transport proteins with a wide tissue distribution have been identified during the last 15 years. Several important examples of their role in drug disposition and drug-drug interactions have been described to date. Investigation of drug-drug interactions at the transport protein level are therefore of increasing interest to the academic, industrial and regulatory research communities. The gene expression of transport proteins involved in drug transport was investigated in the jejunum, liver, kidney and colon to better understand their influence on the ADMET properties of drugs. In addition, the gene and protein expression of transport proteins in cell lines, widely used for predictions of drug transport and metabolism, was examined. The substrate and inhibitor heterogeneity of many transport proteins makes it difficult to foresee whether the transport proteins will cause drug-drug interactions. Therefore, in vitro assays for OCT1 and OATP1B1, among the highest expressed transport proteins in human liver, were developed to allow investigation of the inhibitory patterns of these proteins. These assays were used to investigate two data sets, consisting of 191 and 135 registered drugs and drug-like molecules for the inhibition of OCT1 and OATP1B1, respectively. Numerous new inhibitors of the transport proteins were identified in the data sets and the properties governing inhibition were determined. Further, antidepressant drugs and statins displayed strong inhibition of OCT1 and OATP1B1, respectively. The inhibition data was used to develop predictive in silico models for each of the two transport proteins. The highly polymorphic nature of some transport proteins has been shown to affect drug response and may lead to an increased risk of drug-drug interactions, and therefore, the OCT1 in vitro assay was used to study the effect of common genetic variants of OCT1 on drug inhibition and drug-drug interactions. The results indicated that OCT1 variants with reduced function were more susceptible to inhibition. Further, a drug-drug interaction of potential clinical significance in the genetic OCT1 variant M420del was proposed. In summary, gene expression of transport proteins was investigated in human tissues and cell lines. In vitro assays for two of the highest expressed liver transport proteins were used to identify previously unknown SLC transport protein inhibitors and to develop predictive in silico models, which may detect previously known drug-drug interactions and enable new ones to be identified at the transport protein level. In addition, the effect of genetic variation on inhibition of the OCT1 was investigated.

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In plants, long distance transport of sugars from photosynthetic source leaves to sink organs comprises different crucial steps depending on the species and organ types. Sucrose, the main carbohydrate for long distance transport is synthesized in the mesophyll and then loaded into the phloem. After long distance transport through the phloem vessels, sucrose is finally unloaded towards sink organs. Alternatively, sugar can also be transferred to non‐plant sinks and plant colonization by heterotrophic organisms increases the sink strength and creates an additional sugar demand for the host plant. These sugar fluxes are coordinated by transport systems. Main sugar transporters in plants comprise sucrose (SUTs) and monosaccharide (MSTs) transporters which constitute key components for carbon partitioning at the whole plant level and in interactions with fungi. Although complete SUTs and MSTs gene families have been identified from the reference Dicot Arabidopsis thaliana and Monocot rice (Oriza sativa), sugar transporter families of the leguminous plant Medicago truncatula, which represents a widely used model for studying plant-fungal interactions in arbuscular mycorrhiza (AM), have not yet been investigated.With the recent completion of the M. truncatula genome sequencing as well as the release of transcriptomic databases, monosaccharide and sucrose transporter families of M. truncatula were identified and now comprise 62 MtMSTs and 6 MtSUTs. I focused on the study of the newly identified MtSUTs at a full family scale; phylogenetic analyses showed that the 6 members of the MtSUT family distributed in all three Dicotyledonous SUT clades; they were named upon phylogenetic grouping into particular clades: MtSUT1-1, MtSUT1-2, MtSUT1-3, MtSUT2, MtSUT4-1 and MtSUT4-2. Functional analyses by yeast complementation and expression profiles obtained by quantitative RT-PCR revealed that MtSUT1-1 and MtSUT4-1 are H+/sucrose symporters and represent key members of the MtSUT family. Conservation of transport capacity between orthologous leguminous proteins, expression profiles and subcellular localization compared to previously characterized plant SUTs indicate that MtSUT1-1 is the main protein involved in phloem loading in source leaves whilst MtSUT4-1 mediates vacuolar sucrose export for remobilization of intracellular reserve.The AM symbiosis between plants and fungi from the phylum Glomeromycota is characterized by trophic exchanges between the two partners. The fungus supplies the autotrophic host with nutrients and thereby promotes plant growth. In return, the host plant provides photosynthate (sugars) to the heterotrophic symbiont. Here, sugar fluxes from plant source leaves towards colonized sink roots in the association between the model leguminous plant M. truncatula and the arbuscular mycorrhizal fungus (AMF) Glomus intraradices were investigated. Sugar transporter candidates from both the plant and fungal partners presenting differential expression profiles using available transcriptomic tools were pinpointed. Gene expression profiles of MtSUTs and sugar quantification analyses upon high and low phosphorus nutrient supply and inoculation by the AMF suggest a mycorrhiza-driven stronger sink in AM roots with a fine-tuning regulation of MtSUT gene expression. Conserved regulation patterns were observed for orthologous SUTs in response to colonization by glomeromycotan fungi.In parallel, a non-targeted strategy consisting in the development of a M. truncatula - G. intraradices expression library suitable for yeast functional complementation and screening of symbiotic marker genes, similar to the approach that led to the identification of the first glomeromycotan hexose transporter (GpMST1), has been developed in this study. [...]